WO2021113866A1

WO2021113866A1 - Improved semen processing for in vitro fertilization

Info

Publication number: WO2021113866A1
Application number: PCT/US2020/070843
Authority: WO
Inventors: Rodrigo BOHRER; Bruna CARDOSO
Original assignee: Abs Global, Inc.
Priority date: 2019-12-03
Filing date: 2020-12-03
Publication date: 2021-06-10

Abstract

The present teachings provide for and include methods of preparing semen for in vitro fertilization (IVF). Provided are methods of removing dead cells from semen preparations by applying the semen to a colloidal silica gradient and centrifuging the gradient. In some configurations, the gradient is a 45-90% colloidal silica gradient and the centrifugation is at 419 x g. Further provided are use of the method of the present teachings for processing sex selected semen. Also provided are ova and zygotes matured therefrom that have been fertilized by semen that has been processed by a method of the present teachings.

Description

Improved Semen Processing for In Vitro Fertilization

Cross Reference to Related Application

[0001] This application claims benefit of and priority to US Provisional Application 62/943,030 filed on December 3, 2019. Application 62/943,030 is herein incorporated by reference in its entirety.

Technical Field

[0002] The present disclosure relates to the field of in vitro fertilization. Specifically, this application relates to the processing of semen for use in in vitro fertlization. More specifically, the present disclosure relates to the processing of sex selected semen for in vitro fertilization.

Background

[0003] Livestock in vitro fertilization (IVF) is a multi-million dollar a year business, wherein embryos are created outside of an animal by fertilizing one or more egg cells from a female parent with sperm cells from a male parent. Elite IVF-produced embryos-i.e. where one or both parents have elite genetics — are particularly valuable to farmers because the elite genetics from the parent(s) translates into improved performance characteristics of the livestock animal derived from the elite parents; elite embryos and semen sell for thousands of dollars on the open market. For farmers, utilizing elite IVF-produced embryos can significantly improve production and revenue. The ability to create enough embryos via IVF to populate commercial farming herds is rate-limited by the technical limitations of the in vitro fertilization process. For a company fertilizing 2.5 million oocytes a year, even a 1% gain in efficiency can lead to a $250,000 increase in revenue. This can be especially true for oocytes that have been fertilized with sex- selected semen. In dairy operations, for example, female calves are more valuable than male calves, since male calves cannot produce milk and have limited value as a source of meat; therefore, providing female embryos — i.e. embryos produced using semen that has been sexed to contain primarily X-chromosome-bearing sperm cells, assures a farmer maximum value for their investment.

[0004] However, the sex-selection process makes sex-selected semen more sensitive to stress, and therefore, sex selected semen requires gentler handling during processing than conventional semen. The sexing process inevitably results in the death of some cells, and additional stresses, including but not limited to freezing and thawing. This increased sensitivity to stress increases the proportion of dead cells that are contained in a sexed semen sample. The higher proportion of dead cells can make it difficult to assess the number or proportion of fertilization-competent sperm cells in a sample, can lead to mistakes in determining the concentrations needed for IVF, and can otherwise interfere with the process.

[0005] Dead cells are not typically removed from conventional semen when used for artificial insemination. When conventional semen is used for IVF, dead cells are often removed using magnetic separation (e.g. MACS), by using the live cells’ gravitropism to purify them using a swim up process, or by density gradient centrifugation through a colloidal solution of silica coated with polyvinylpyrrolidone (PVP).

[0006] Prior methods of removing dead cells rely on particular gradient parameters, and speeds and durations of centrifugation. For example, Parrish et al. (Parrish, J.J., et al., Theriogenology., 1995, 44, 859-869) disclosed using a 45% and 90% gradient of colloidal silica centrifuged at 700 x g for 15 minutes; Tanphaichitr et al. (Tanphaichitr, N., et al., Gamete Res., 1988, 20, 67-81) disclosed the use of a 90%-47% gradient of colloidal silica spun at 600 x g for 30 minutes to clean up human sperm; Matas et al. (Matas, C., Anim. Reprod. Sci., 2011, 127, 62- 72) disclosed the use of a 45% - 90% gradient of colloidal silica centrifuged at 700 x g for 30 minutes to clean up porcine sperm; and Noguchi et al. (Noguchi, N., et al., Zygote, 2015, 23, 68- 75) disclosed the use of a 50%-80% gradient of colloidal silica and centrifuging at 700 x g for 20 minutes to clean up freeze-thawed porcine semen. However, none of these approaches is suitable for commercial IVF applications because cells prepared through these processes do not provide a sufficient number of actively motile, unstressed, and fertility competent cells, and none of them disclose centrifugation at low speeds, or involve the use of sexed semen.

[0007] Because the use of semen for IVF requires a sufficient number of sperm cells that are actively motile, and unstressed, gentler methods of cleaning semen are required.

Summary

[0008] The present inventors have developed superior methods of preparing livestock semen for use in IVF. These methods increase the efficiency of the procedures and lead to an increase in the number of embryos produced, and gains in commercial production and revenue.

[0009] In some embodiments, the present teachings can provide for and include a method of preparing semen for in vitro fertilization comprising: providing semen comprising sperm cells; providing a 45%-90% gradient of colloidal silica; depositing the semen on top of the gradient of colloidal silica; and centrifuging the semen and gradient at a speed and for a duration sufficient to produce a supernatant and a pellet comprising sperm cells. In various configurations, the speed of centrifugation is sufficient to exert a force of 400-450 x g. In various configurations, the semen and gradient can be centrifuged for 10 minutes. In various configurations, the semen and gradient can be centrifuged at about 419 x g. In various configurations, the semen and gradient can be centrifuged at 419 x g for 10 minutes. In various configurations, the semen can be sex- selected semen. In some configurations, the sex-selected semen can be selected using laser ablation. In various configurations, the semen can be livestock semen. In various configurations, the semen can be bovine semen. In various configurations, the semen can be porcine semen. In various configurations, the semen can be Sus scrofa domesticus semen. In various configurations, the semen can be Bos taurus semen. In various configurations, the semen can be Bos indicus semen. In various configurations, the semen can be sex-selected Bos taurus semen, sex-selected Bos indicus semen, or sex-selected Sus scrofa domesticus semen. In various configurations, wherein the colloidal silica can be coated with polyvinylpyrrolidone.

[0010] In various configurations, the centrifugation can form a supernatant and a pellet, wherein the pellet is comprised of sperm cells. The method can further comprise: removing the supernatant and collecting the sperm cells that comprise the pellet. In some configurations, the method of the present teachings can further comprise contacting the collected sperm cells that had comprised the pellet with at least one ovum whereby at least one of the sperm cells fertilizes the at least one ovum.

[0011] In various configurations of the present teachings described supra , the livestock semen can be conventional semen, that is, semen that has not been subjected to a sex selection process. In some configurations, semen may be raw ejaculate. In other configurations, the semen may include one or more of: an extender, a cryopreservative, or an antioxidant.

[0012] In various configurations of the present teachings described supra , the livestock semen can be sex-selected semen. The sex-selected semen can be produced from raw ejaculate, or from semen that includes an extender, a cryopreservative, or an antioxidant. In some configurations, the sex-selected semen can be sex-selected using laser ablation. In various configurations, the sex-selected semen can be sex-selected using a geometric focusing fluid cytometry chip. In various configurations, the sex-selected semen can be sex-selected using a microfluidic chip. [0013] In various embodiments, the present teachings provide for and include a zygote or embryo that matures from an ovum fertilized in accordance with the present teachings.

[0014] In various embodiments, the present teachings provide for and include an animal that matures from an ovum fertilized in accordance with the present teachings. In some configurations, the animal can be a Bos taurus animal. In various configurations, the animal can be a Sus scrofa domesticus animal. [0015] In some embodiments, the present teachings provide for and include a method of preparing semen for in vitro fertilization comprising: providing semen; providing a 45%-90% gradient of colloidal silica; depositing the semen on top of the gradient of colloidal silica; centrifuging the semen and the gradient at a speed and for a duration sufficient to form a supernatant and a pellet comprising sperm cells; removing the supernatant; washing the pellet; collecting the sperm cells that comprise the pellet; and contacting at least one ova with the sperm cells, whereby at least one of said sperm cells fertilizes the at least one ovum. In some configurations, the semen can be Bos taurus semen. In some configurations, the semen can be Bos indicus semen. In various configurations, the semen can be Sus scrofa domesticus semen. [0016] In some embodiments, the present teachings provide for and include a method of preparing semen for in vitro fertilization comprising: providing semen; providing a 45%-90% gradient of colloidal silica; depositing the semen on top of the gradient of colloidal silica; centrifuging the gradient at a speed and for a duration sufficient to form a supernatant and a pellet comprising sperm cells; removing the supernatant; washing the pellet; collecting the sperm cells that comprised the pellet; and contacting at least one ova with the sperm cells, whereby at least one sperm cell fertilizes the at least one ovum. In some configurations, the semen may be sex-selected semen. In further configurations, the sex-selected semen has been selected by laser ablating the non-selected cells. The sexed semen produced by laser ablation may include the ablated cells, and cell debris resulting therefrom. In some configurations, the semen can be Bos taurus semen. In some configurations, the semen can be Bos indicus semen. In various configurations, the semen can be Sus scrofa domesticus semen.

[0017] In various embodiments, the semen used in these processes can be sexed semen.

Sexed semen can be prepared by a variety of techniques known in the art, including but not limited to droplet sorting, fluid switching, laser steering or trapping, photonic pressure, acoustic wave sorting, and laser ablation. Sexed semen produced by any of these techniques can be either primarily X-chromosome-bearing (X-skew) or primarily Y-chromosome-bearing (Y-skew). [0018] In various embodiments, the present teachings include a straw of semen prepared according to any of the foregoing.

Description

[0019] In some embodiments, the present teachings can provide for and include a method of preparing semen for in vitro fertilization comprising: providing semen comprising sperm cells; providing a 45%-90% gradient of colloidal silica; depositing the semen on top of the gradient of colloidal silica; and centrifuging the semen and gradient at a speed and for a duration sufficient to produce a supernatant and a pellet comprising sperm cells. In various configurations, the speed of centrifugation will be sufficient to exert a force on the semen and gradient of 400-450 x g. Skilled artisans will appreciate that adjustment of RPM will be necessary when changing centrifuges or centrifuge rotors, but by maintaining the force at 400-450 x g, a skilled artisan can achieve like results in different centrifuges. In various embodiments, the centrifugation can be at 410-440 x g. In various embodiments the centrifugation can be at 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, or 450 x g. In some configurations, the centrifugation can be at 419 x g. In various configurations, the centrifugation can be about 419 x g. In some configurations, the centrifugation can be about 420 x g. In various configurations, the centrifugation can be 5-15 minutes. In some configurations, the centrifugation can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes.

[0020] As discussed herein, a gradient designated by a dash indicates two concentrations of a separating agent that have been layered on top of each other. For example, a 45%-90% gradient can have a 45% layer and a 90% layer. The colloidal silica gradient can have an about 45% layer and an about 90% layer. The colloidal silica gradient can have a 45% layer and a 90% layer. The colloidal silica gradient can have one or more layers of intermediate concentrations interposed between the 45% layer and the 90% layer; for example the colloidal silica gradient can have a 45% layer (or an about 45% layer) and a 90% layer (or an about 90% layer) and a 70 % layer (or and about 70% layer) interposed between the other two layers. While a precise gradient is required, skilled artisans will appreciate that a variance of about 5-10% in each concentration will produce like, if less efficient, results.

[0021] Artificial insemination (AI) and in vitro fertilization (IVF) technology are well established in the art of livestock breeding. Semen and ova can be harvested for use in the present teachings via any method known in the art. These techniques are especially well developed for bovine and porcine semen. Semen is prepared for preservation by adding extenders and other cryopreservation agents known in the art. Common species of livestock that are bred using AI and IVF include, such as and without limitation, Bos taurus , Bos indicus , Sus scrofa domesticus , Equus cabal lus, and Ovis aries.

[0022] The present teachings provide for and include methods of preparing sex selected semen for in vitro fertilization. As discussed supra , there are several methods of sex selection of livestock semen known in the art. Commonly applied techniques include droplet sorting, fluid switching, laser steering or trapping, photonic pressure, and acoustic wave sorting. One preferred method is flowing semen through a microfluidic chip, differentiating X-chromosome-bearing sperm cells from Y-chromosome-bearing sperm cells, and then laser ablating the undesired cells. This procedure can be performed using apparatuses and methods described in U.S. Patents 8,961,904 and 10,069,027. Laser ablation of undesired cells is described in U.S. Pat. Nos. 8,933,395, 9,000,357, 9,140,690, and 9,335,295. Where the semen for preparation according to the present teachings is sex-selected semen, said sex selected semen may include both intact, progressively motile and/or fertilization competent sperm cells, and dead, inactivated, and/or fertilization incompetent sperm cells. In certain configurations where the semen is sex-selected semen prepared using laser ablation, the sex-selected semen may include both intact, unablated sperm cells, and sperm cells that have been killed or inactivated by an ablation laser.

[0023] Methods of culturing in vitro fertilized ova and implanting them into surrogate dams for gestation until birth are also known in the art. Various techniques related to IVF are described in U.S. Patent Application Publication No. US20180352808.

[0024] Colloidal silica is sold commercially from various suppliers. Colloidal silica coated with polyvinylpyrrolidone (PVP) is sold under the trade name PERCOLL™ (GE Healthcare Company, Marlborough, MA). Colloidal silica coated with silane is sold under the tradename PERCOLL™ Plus. Skilled persons will realize that many substitutions are possible to achieve similar results.

Examples

[0025] The present teachings including descriptions provided in the Examples that are not intended to limit the scope of any claim or embodiment. Unless specifically presented in the past tense, an example can be a prophetic example or an actual example. The following non-limiting examples are provided to further illustrate the present teachings. Those of skill in the art, in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.

Example 1

[0026] This example illustrates a semen preparation method of the present teachings.

[0027] IVF ready media was prepared by supplementing commercial IVF Media with heparin, PHE, and antibiotic according to standard protocols. Colloidal silica coated with polyvinylpyrrolidone (PVP) was filtered through a 22 mM filter to form 90% colloidal silica. For each sex-selected bovine semen sample, 225 mΐ of this 90% colloidal silica was added to a 1.5 ml microtube. 225 mΐ of IVF-TL was then added to the tube and the two substances were mixed to form 45% colloidal silica. 450 mΐ of 90% colloidal silica was then added to the bottom of the tube to form a 45%-90% colloidal silica gradient. Straws of semen were then thawed and the thawed semen was added to the top (45% colloidal silica) layer of the colloidal silica gradient. The tubes were then spun at 419 x g for 10 minutes. The supernatant was removed and the pellet comprising the sperm was retained and resuspended in 1 ml of “IVF Ready” media. The tubes were then spun at 194 x g for 5 minutes, and the pellet transferred to a pre-warmed 0.5 ml microfuge tube. The pellet was then resuspended and the semen used for IVF according to standard protocols.

Example 2

[0028] This example illustrates comparative data of sex-selected semen prepared under standard protocols verses sex-selected semen prepared using a method of the present teachings. [0029] X-skewed sex selected semen samples from 8 Bos taurus bulls were aliquoted and half of the samples were prepared as described in Example 1. The other half were prepared according to standard protocols — this protocol is similar to that described in Example 1, with the following modifications: the colloidal silica gradient was a 78% to 22% gradient, the first centrifugation was at 6700 x g for five minutes, and the second centrifugation was at 1100 x g for 3 minutes. Each sample was then used for IVF. Results are depicted in Tables 1 and 2.

Table 1 IVF Overall Results

[0030] of the present teachings produce 200 more embryos from 200 fewer oocytes, illustrating that the method of the present teachings is more efficient than conventional methods.

Table 2 IVF Statistics

[0031] IVC refers to In Vitro Cultured oocytes, which is the number of oocytes still alive after fertilization. The above numbers illustrate that more fertilized oocytes become embryos using a protocol of the present teachings compared to the previous standard protocol.

[0032] Example 3

[0033] This example illustrates comparative data between results for pre-IVF clean-up of semen between a method of the present teachings and a conventional method.

[0034] X-skewed sex selected semen samples from 14 Bos taurus bulls were aliquoted and half of the samples were prepared as described in Example 1. The other half were prepared according to standard protocols — this protocol is similar to that described in Example 1, with the following modifications: the colloidal silica gradient was a 78% to 22% gradient, the first centrifugation was at 6700 x g for 5 minutes, and the second centrifugation was at 1100 x g for 3 minutes. Each sample was then used for IVF. Results are depicted in Tables 3 and 4.

Table 3- IVF Results

Table 4 - IVF Statistics

higher percentage of oocytes that formed embryos relative to the control group. Example 4

[0036] This example illustrates a semen preparation according to the present teachings. [0037] IVF ready media is prepared by supplementing commercial IVF Media with heparin, PHE, and antibiotic according to standard protocols. Colloidal silica coated with polyvinylpyrrolidone (PVP) is filtered through a 22 mM filter to form 90% colloidal silica. For each Bos indicus semen sample, 225 mΐ of this 90% colloidal silica is added to a 1.5 ml microtube. 225 mΐ of IVF-TL is then added to the tube and the two substances are mixed to form 45% colloidal silica. 450 mΐ of 90% colloidal silica are then added to the bottom of the tube to form a 45%-90% colloidal silica gradient. Straws of semen are then thawed and the thawed semen is added to the top (45% colloidal silica) layer of the colloidal silica gradient. The tubes are then spun at 425 x g for 10 minutes. The supernatant is removed and the pellet comprising the sperm is retained and resuspended in 1 ml of “IVF Ready” media. The tubes are then spun at 194 x g for 5 minutes, and the pellet transferred to a pre-warmed 0.5 ml microfuge tube. The pellet is then resuspended and the is semen used for IVF according to standard protocols. Example 5

[0038] This example illustrates a semen preparation according to the present teachings.

[0039] IVF ready media is prepared by supplementing commercial IVF Media with heparin, PHE, and antibiotic according to standard protocols. Colloidal silica coated with polyvinylpyrrolidone (PVP) is filtered through a 22 mM filter to form 90% colloidal silica. For each Sus scrofa domesticus semen sample, 225 mΐ of this 90% colloidal silica is added to a 1.5 ml microtube. 225 mΐ of IVF-TL is then added to the tube and the two substances are mixed to form 45% colloidal silica. 450 mΐ of 90% colloidal silica are then added to the bottom of the tube to form a 45%-90% colloidal silica gradient. Semen is removed from a fresh straw and is added to the top (45% colloidal silica) layer of the colloidal silica gradient. The tubes are then spun at 410 x g for 10 minutes. The supernatant is removed and the pellet comprising the sperm is retained and resuspended in 1 ml of “IVF Ready” media. The tubes are then spun at 194 x g for 5 minutes, and the pellet transferred to a pre-warmed 0.5 ml microfuge tube. The pellet is then resuspended and the is semen used for IVF according to standard protocols.

Example 6

[0040] This example illustrates a semen preparation according to the present teachings.

[0041] IVF ready media is prepared by supplementing commercial IVF Media with heparin, PHE, and antibiotic according to standard protocols. Colloidal silica coated with polyvinylpyrrolidone (PVP) is filtered through a 22 mM filter to form 90% colloidal silica. For each Y-skewed sex-selected Sus scrofa domesticus semen sample, 225 mΐ of this 90% colloidal silica is added to a 1.5 ml microtube. 225 mΐ of IVF-TL is then added to the tube and the two substances are mixed to form 45% colloidal silica. 450 mΐ of 90% colloidal silica are then added to the bottom of the tube to form a 45%-90% colloidal silica gradient. Semen is removed from a fresh straw and is added to the top (45% colloidal silica) layer of the colloidal silica gradient.

The tubes are then spun at 410 x g for 10 minutes. The supernatant is removed and the pellet comprising the sperm is retained and resuspended in 1 ml of “IVF Ready” media. The tubes are then spun at 194 x g for 5 minutes, and the pellet transferred to a pre-warmed 0.5 ml microfuge tube. The pellet is then resuspended and the is semen used for IVF according to standard protocols.

[0042] All publications cited herein are hereby incorporated by reference, each in their entirety.

Claims

What is claimed is:

1. A method of preparing semen for in vitro fertilization comprising: providing semen comprising sperm cells; providing a 45%-90% colloidal silica gradient; depositing the semen on top of the colloidal silica; and centrifuging the semen and gradient at 400-450 x g.

2. A method of preparing semen according to claim 1, wherein the gradient is centrifuged for 10 minutes.

3. A method of preparing semen according to claim 1, wherein the gradient is centrifuged at about 419 x g.

4. A method of preparing semen according to claim 1, wherein the gradient is centrifuged at 419 x g for 10 minutes.

5. A method of preparing semen according to claim 1, wherein the semen is bovine semen.

6. A method of preparing semen according to claim 1, wherein the semen is porcine semen.

7. A method of preparing semen according to claim 1, wherein the semen is selected from the group consisting of Sus scrofa domesticus semen and Bos taurus semen.

8. A method of preparing semen according to claim 1, wherein the colloidal silica is coated with polyvinylpyrrolidone.

9. A method of preparing semen according to claim 1, wherein the centrifugation forms a supernatant and a pellet comprising sperm cells and wherein the method further comprises: removing the supernatant; and collecting the sperm cells comprising the pellet.

10. A method of preparing semen according to claim 9, further comprising contacting at least one ovum with the collected sperm cells, whereby at least one sperm cell fertilizes the at least one ovum.

11. A method according to method any of the previous claims wherein said semen is sex-selected semen. using laser ablation.

13. The method of claim 12, wherein said sex-selected semen comprises both intact, unablated cells and ablated cells that have been inactivated or killed.

14. A method of preparing sex-selected semen according to claim 11, wherein the sex-selected semen is sex-selected using a microfluidic chip.

15. A zygote or embryo that matures from an ovum fertilized in accordance with any of claims claim 10-14.

16. An animal that matures from an ovum fertilized according to any one of claims 10-14.

17. An animal in accordance with claim 16, wherein the animal is a Bos taurus animal.

18. An animal in accordance with claim 16, wherein the animal is a Sus scrofa domesticus animal.

19. A method of preparing sex-selected semen for in vitro fertilization comprising: providing sex-selected semen that was selected using a microfluidic chip and laser ablating the non-selected cells; providing a 45%-90% colloidal silica gradient; depositing the sex-selected semen on top of the colloidal silica gradient; centrifuging the gradient at 419 x g for 10 minutes to form a supernatant and a pellet comprising sperm cells; removing the supernatant; washing the pellet; collecting the sperm cells that comprised the pellet; and contacting at least one ova with the collected sperm cells, whereby at least one sperm cell fertilizes the at least one ovum.

20. A method in accordance with claim 19, wherein the semen is selected from the group consisting of Bos taurus semen and Sus scrofa domesticus semen.